Parathyroid hormone (PTH) is a principal regulator of bone and mineral-ion homeostasis. When given exogenously, PTH can increase or decrease bone mass, depending upon the dose and temporal pattern of administration. Once-daily injected PTH uniquely augments osteoblastic bone formation and is the only anabolic therapy currently approved for the treatment of osteoporosis. Osteoblasts (Obs), the main targets of PTH action in bone, express PTH/PTHrP receptors (PTHRIs) which can activate, in parallel, multiple effector pathways, including cyclic AMP/protein kinase A, phospholipase C (PLC)-dependent protein kinase C(s) (PKCs) and PLC-independent PKC(s). Activation of these pathways via normal PTHRIs can be separated by use of novel mutant signal-selective PTH analogs. Using such analogs, we have shown that PLC-independent signaling mechanism(s) play a positive modulatory role in the anabolic action of PTH in normal mice and that PKC6 is activated by PTHRIs via a PLC-independent mechanism that contributes to PTH stimulation of Ob differentiation in vitro. Further, mice lacking PKC6 have reduced bone mass and develop fractures on a low-calcium diet. In other preliminary studies we also found that the transcriptional co-regulator protein CITED1 is up-regulated in Obs following intermittent but not continuous PTH exposure. CITEDI-knockout (KO) Obs in vitro show increased basal differentiation and strikingly augmented responsiveness to cAMP-dependent cyclic PTH stimulation of Ob differentiation. Using PKC6 and CITED1 KO mice, we will address the following hypotheses: (I) that PKC6 mediates positive modulatory effects of cAMP- and PLC-independent PTHR1 signaling on the anabolic PTH response and may constrain the catabolic action of continuous PTH; and (II) that CITED1 induction by intermittent PTH may feed back to preferentially suppress cAMP-dependent responses and limit the anabolic effect of PTH. Responses of bone in KO mice to intermittent vs. continuous PTH treatment will be assessed by densitometric, microstructural, mechanical, histological, histomorphometric and molecular (mRNA) analyses, supplemented by ex vivo and in vitro assays of Ob commitment, differentiation, proliferation and apoptosis as well as of osteoclast formation and activity. Molecular mechanisms of PKC6 activation and action and of CITED1 action will be defined using established Ob cell lines and calvarial Obs from KO mice. This work will contribute important new insight into how the multiple signals triggered by PTH in target cells of bone are integrated to increase or reduce bone mass. Information gained could guide design of new forms of PTH with selective signaling properties and/or of adjunctive agents directed at PKC5 or CITED1 to augment the therapeutic effect of PTH in osteoporosis.